Peripheral nerve grafts (PNGs) provide an excellent substratum for axonal regrowth;they can direct regenerating axons towards a specific target and they facilitate electrophysiological experimentation to detect synaptic connectivity between regenerating axons and distal spinal cord neurons. A major impediment to this and all other transplantation approaches after spinal cord injury is the poor growth of axons out of the graft back into the host spinal cord. We have combined Chondroitinase (ChABC, to digest inhibitory chondroitin sulfate proteoglycan molecules) with PN grafting in rats and have anatomical and electrophysiological evidence for functional synapse formation by injured, regenerating axons in both acute and delayed (chronic injury) treatment paradigms. Recently we replicated this rat acute PNG approach in cats where we observed thousands of axons regenerating into the graft, a small percentage of which extended from the graft into the spinal cord distal to the injury, and spinal neurons synaptically activated (determined by c-Fos immunoreactivity) after electrical stimulation of the nerve graft. While we will continue to use rat models for expanding our treatment repertoire, the objective of this study is to focus on application of our treatment strategies to chronically injured cats as a necessary preclinical step before translation into human research. The cat model permits us to investigate issues related to the scaling up of a transplantation model, cats are easily trained to perform locomotor tasks, and recovery of function can be assessed by kinematic and electrophysiological measures. The biomechanics of locomotion are better defined in cats and cats have a hindlimb gait that is close to human than is the rat. The proposed work also will provide information about the ability to effectively treat glial scarring in a large animal, the ability to promote structural and functional regeneration in a large animal with a chronic injury and the potential for rehabilitation training to foster regeneration and functional recovery. There are 2 Specific Aims for this project. 1) We will identify the source and extent of axonal regeneration into a PNG after chronic injury and test whether these axons form functional connections across the lesion. 2) We will test whether the start time of physical rehabilitation affects outgrowth, integration and/or synaptic activity of regenerating axons. A combination of treatment strategies will be used, including transplantation, ChABC treatments and treadmill training to promote activity dependent plasticity. Structural repair will be assessed by anatomical tract tracing and immunocytochemical labeling;forelimb-hindlimb coordination will be assessed by kinematic and electromyogram (EMG) analysis;functional reconnection will be measured during electrophysiological stimulation of the graft and by c-fos expression in synaptically activated neurons. Surgical intervention after SCI usually is not an option until the patient is stabilized, thus the majority of individuals with SCI likely will be chronically injured before a treatment strategy for repair is initiated. Our work with chronically injured rats demonstrates the ability to promote long distance regeneration with formation of functionally active synapses distal to an injury. The proposed study will take advantage of the treatment approaches that have been (and are being) developed with chronically injured rats, but will apply them to a large animal model of SCI. This preclinical advancement is a crucial step towards translation to a clinical application. We propose a unique approach to address a very important aspect of SCI, i.e. chronic injury in a large animal model. Locomotor training of injured cats has been carried out by numerous labs, but not in a situation where axon regeneration is facilitated. This will be a novel application of neuroregeneration and neurorehabilitation techniques to increase our understanding of the potential for repair after SCI. PUBLIC HEALTH RELEVANCE: Different transplantation models have been used to demonstrate that under the right conditions neurons in spinal cord injured adult rats will regenerate their nerve processes (axons) into and through the transplant, forming a bridge across the lesion to restore lines of communication between the brain and spinal cord. Part of the success that has been demonstrated with these models involves additional treatment of the spinal cord tissue adjacent to the injury, to either increase the presence of growth promoting molecules or to decrease the presence of growth inhibitory molecules. An important issue to address concerns the time after injury when a treatment might be most effective and we have explored this question by delaying treatment for weeks to months after injury. We have demonstrated that chronically injured neurons in rats retain the capacity for regeneration for long (at least 12 months) post injury periods. This observation directly impacts the overwhelming number of spinal cord injured patients because of the perception that most surgical interventions should be delayed until the individual is stable and opportunities for spontaneous recovery have subsided. As a prelude to work with human patients we have pursued our peripheral nerve graft studies in a larger animal model, spinal cord injured cats. The purpose of this study was to determine if multiple surgical procedures could be performed on the cat spinal cord without causing significant pain or discomfort to the animal and if peripheral nerve grafts used to bridge the lesion in rats would be equally effective in cats, providing a channel for regenerating axons to form functional synaptic contacts with spinal cord neurons. We have data demonstrating the successful application of our combination of treatments to the adult cat spinal cord when carried out immediately after injury. The present proposal will extend this observation to the chronically injured cat model where treatment strategies will be delayed for several months, with the objective of advancing the use of a combination of treatment strategies towards translation into human application. The proposed work will provide valuable information about the ability to perform surgical reconstruction of spinal cord circuitry in a large animal, to determine if size might be a hindrance to successful regeneration. It also will provide an understanding of the potential to promote structural and functional recovery in a large animal with a longstanding (chronic) injury and of the ability to foster greater recovery through aggressive physical rehabilitation training. We feel that results of this study would have direct clinical relevance with the major risk being in not pursuing this line of investigation.